Method for semi-automated microscopy of filtration-enriched circulating tumor cells

BACKGROUND: Circulating tumor cell (CTC)-filtration methods capture high numbers of CTCs in non-small-cell lung cancer (NSCLC) and metastatic prostate cancer (mPCa) patients, and hold promise as a non-invasive technique for treatment selection and disease monitoring. However filters have drawbacks that make the automation of microscopy challenging. We report the semi-automated microscopy method we developed to analyze filtration-enriched CTCs from NSCLC and mPCa patients. METHODS: Spiked cell lines in normal blood and CTCs were enriched by ISET (isolation by size of epithelial tumor cells). Fluorescent staining was carried out using epithelial (pan-cytokeratins, EpCAM), mesenchymal (vimentin, N-cadherin), leukocyte (CD45) markers and DAPI. Cytomorphological staining was carried out with Mayer-Hemalun or Diff-Quik. ALK-, ROS1-, ERG-rearrangement were detected by filter-adapted-FISH (FA-FISH). Microscopy was carried out using an Ariol scanner. RESULTS: Two combined assays were developed. The first assay sequentially combined four-color fluorescent staining, scanning, automated selection of CD45(−) cells, cytomorphological staining, then scanning and analysis of CD45(−) cell phenotypical and cytomorphological characteristics. CD45(−) cell selection was based on DAPI and CD45 intensity, and a nuclear area >55 μm(2). The second assay sequentially combined fluorescent staining, automated selection of CD45(−) cells, FISH scanning on CD45(−) cells, then analysis of CD45(−) cell FISH signals. Specific scanning parameters were developed to deal with the uneven surface of filters and CTC characteristics. Thirty z-stacks spaced 0.6 μm apart were defined as the optimal setting, scanning 82 %, 91 %, and 95 % of CTCs in ALK-, ROS1-, and ERG-rearranged patients respectively. A multi-exposure protocol consisting of three separate exposure times for green and red fluorochromes was optimized to analyze the intensity, size and thickness of FISH signals. CONCLUSIONS: The semi-automated microscopy method reported here increases the feasibility and reliability of filtration-enriched CTC assays and can help progress towards their validation and translation to the clinic. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12885-016-2461-4) contains supplementary material, which is available to authorized users

Additional file 3: Table S2. of Method for semi-automated microscopy of filtration-enriched circulating tumor cells

FISH spots per CTC of a patient with an ERG-rearranged tumor depending on the step. (DOC 63 kb

Additional file 1: Figure S1. of Method for semi-automated microscopy of filtration-enriched circulating tumor cells

Examples of gene rearrangement and gain/amplification detection in filtration enriched-cell lines by filter-adapted-FISH (FA-FISH). (A) Example of gene rearrangement detection. (B) Example of gain/amplification detection. Scale: white bars = 10 μm. (TIF 8523 kb

Additional file 2: Table S1. of Method for semi-automated microscopy of filtration-enriched circulating tumor cells

FISH spots detected per CTC in a patient with a ROS1-rearranged tumor depending on the step. (DOC 68 kb